Abstract By use of an antiserum raised against conjugated ovine corticotropin releasing factor (CRF 1–41), nerve fibres can be stained immunocytochemically in the external zone of the median eminence of rats. The presence of CRF-immunoreactive (CRF i) nerve fibres and the plasma corticosterone response to ether stress were studied in rats 6–7 days after making various types of lesions in the hypothalamus. Complete anterolateral deafferentation of the mediobasal hypothalamus caused complete disappearance of CRF i fibres from the median eminence and blocked the corticosterone response to stress. Incomplete anterolateral hypothalamic deafferentation did not prevent the stress-induced increase of corticosterone and in these rats, part of the CRF i nerve fibres remained intact. A horizontal cut placed ventral to the paraventricular nuclei, completely prevented the corticosterone response in those rats that showed a complete disappearance of CRF i nerve fibres from the median eminence. Some rats however, still exhibited CRF i nerve fibres and these animals responded to stress with increased corticosterone levels. A similar horizontal cut made just dorsal to the paraventricular nuclei affected neither the corticosterone response to stress nor the appearance of CRF i nerve fibres in the median eminence. We conclude that the presence of CRF i nerve fibres in the median eminence is a prerequisite for rats to show a pituitary-adrenal response to ether stress and therefore represents the first functional evidence for the role of these hypothalamic CRF i-neurons.